Chikungunya virus susceptibility increases in the absence of mk2b and mk3 genes in zebrafish model

Chikungunya virus (CHIKV) infection imposes a significant socio-economic burden due to the absence of effective antiviral treatments and vaccines. Host factors are essential for the CHIKV life cycle, making them promising targets for antiviral therapy. Previous studies have identified Mitogen-activated protein kinase activated protein kinase 2 (MK2) and Mitogen-activated protein kinase activated protein kinase 3 (MK3) as key host factors in CHIKV infection; however, their role in an animal model remain unclear. This study highlights the critical roles of MK2 and MK3 host factors in an animal model following CHIKV infection. To investigate their functions, mk2b (mk2b-/-), mk3 (mk3-/-), and mk2b-mk3 (mk2b-/-mk3-/-) double knockout zebrafish were generated using the CRISPR-Cas9 technique. A significant high viral titer of CHIKV was observed in case of all knockout groups compared to the wild-type (WT) control using plaque assay, RT-qPCR and immunofluorescence assay. Among the knockout groups, mk3-/- displayed the highest susceptibility to CHIKV, followed by mk2b-/-. In contrast, the mk2b-/-mk3-/- double knockout exhibited the lowest susceptibility to CHIKV infection. Additionally, severe symptoms such as bent body, impaired response to physical stimuli, and increased mortality were most pronounced in mk3-/- larvae compared to other knockouts and the WT. The expression levels of inf{phi}1 and rsad2 were also elevated in all knockout groups during the early days of infection indicating higher interferon response in the absence of mk2b and mk3 during CHIKV infection. In conclusion, this study confirms that the mk2b and mk3 host proteins are essential in controlling the CHIKV infection in organism level and subsequently may contribute in designing antiviral therapeutics in future. Furthermore, the knockout model of mk2b and mk3 in zebrafish could serve as a valuable tool for studying their roles in other viral infections. Author SummaryCHIKV, transmitted by Aedes aegypti and Aedes albopictus mosquitoes, causes febrile illness and has spread across Africa, Asia, Europe, and America. Despite extensive research, effective antiviral drugs and vaccines are yet to be commercially available. This study examines the role of mk2b and mk3 host factors following CHIKV infection. For this purpose, mk2b and mk3 single as well as double knockout have been generated in zebrafish using the CRISPR-Cas-9 technique. Findings suggest that zebrafish exhibit high CHIKV susceptibility in the absence of mk2b and mk3, confirming its importance during infection. Moreover, these three knockout models could serve as a valuable platform for examine the role of mk2b and mk3 in the presence of other viral infection.